P1228: GENOME-WIDE CRISPR-CAS9 SCREENING IDENTIFIES THERAPEUTIC TARGETS FOR MATURE T-CELL MALIGNANCIES
Authors: Sanna Timonen, Vanessa Rebecca Gasparini, Ella Piekkari, Olli Dufva, Shady Awad, Yevhen Akimov, Jana von Jan, Till Braun, Aleksandr Ianevski, Matti Kankainen, Jay Klievink, Marco Herling, Tero Aittokallio, Satu Mustjoki
Journal: HemaSphere (2023)

Topic: 20. Lymphoma Biology & Translational Research Background: Mature T-cell leukemias/lymphomas (MTCL) are rare and mostly aggressive malignancies. Their rarity has contributed to an underrepresentation of specific clinical trials and of approved therapeutic options. Patients with MTCL are predominantly treated with strategies developed for B-cell neoplasms, often leading to disappointing rates of responses and survival. Aims: This study aimed to identify genetic mechanisms in MTCL, which confer drug resistance and sensitivity for selected, clinically relevant anti-cancer drugs. Methods: We first screened 17 drugs in 15 samples from patients with T-prolymphocytic leukemia (T-PLL, the most frequent subgroup of mature T-cell leukemias in Western countries). Idasanutlin, romidepsin, dinaciclib, venetoclax, navitoclax, ruxolitinib, idarubicin, cladribine, and belinostat were effective in 15-100% of these T-PLL samples. To investigate the loss-of-function mutations leading to resistance or sensitivity to these selected drugs, we next performed genome-wide CRISPR-Cas9 knockout screens combined with the drug treatments. To select relevant cell line models, we first tested drug sensitivities in six T-cell leukemia/lymphoma cell lines, and Mac2a resembled best the drug responses seen in T-PLL samples and was therefore selected for the CRISPR screen. Molt4 with engineered TCL1A over-expression to mimic T-PLL was further selected and showed sensitivity to five of the nine tested drugs. After Brunello library transduction and puromycin selection, Mac2a and Molt4-TCL1A cells were treated with the nine selected drugs for up to two weeks at concentrations that kept the overall cell number near-constant. Guide RNA amplicons were amplified and sequenced and the MAGeCK tool was used to identify essential genes needed for cell survival (DMSO-treatment vs. plasmid library) and genes that confer drug resistance or sensitivity (drug- vs. DMSO-treatment). Results: In the STRING database, the essential genes in both cell lines (997 in Mac2a, 173 in Molt4-TCL1A, FDR <0.05) shared common KEGG pathways, such as those related to ribosome (82 in Mac2a, 38 in Molt4-TCL1A) and spliceosome (54 in Mac2a, 16 in Molt4-TCL1A). In addition, 41 oxidative phosphorylation- associated genes were essential in Mac2a only. In drug-treated Molt4-TCL1A, we identified a total of 85 positive selection (resistance) genes, and only a single negative selection (sensitivity) gene (FDR <0.05). In drug-treated Mac2a, a total of 93 genes were identified (52 positive and 43 negative selection hits, FDR <0.05, two genes were both). When combining STRING results from all the hit genes in both cell lines (n=172), p53 signaling turned out as the most significant KEGG pathway (FDR <5.03e-08), with 11 genes (e.g. TP53, TP73, MDM4) found important for both cell lines and responses to all drugs except for romidepsin. Furthermore, eight genes were related to ferroptosis or glutathione metabolism; these were also found important for both cell lines and for all the drugs except for dinaciclib and ruxolitinib. For example, knocking out a ferroptosis repressor gene GPX4 made Mac2a more sensitive to belinostat (FDR <0.017). Summary/Conclusion: A genome-wide CRISPR-Cas9 screen identified genes mediating individual responses to drugs preselected from T-PLL cases in cell line models of Mac2a and Molt4-TCL1A. Mainly p53 signaling and ferroptosis-related genes were associated with responses to most of the tested drugs. As ferroptosis is a process that can be targeted with investigational drugs, it may provide therapeutic options and new drug combinations for mature T-cell malignancies. Keywords: T cell leukemia, T cell lymphoma, Drug sensitivity, Drug resistance